Reverse Genetic Analysis of Antiviral Resistance Mechanisms

抗病毒耐药机制的反向遗传分析

• 批准号: 7202834
• 负责人: Shizuo Akira
• 金额: $ 41.45万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7202834
• 关键词: Japanese encephalitis virus; Newcastle disease virus; biological signal transduction; encephalomyocarditis virus; expression cloning; gene expression profiling; gene targeting; genetic susceptibility; genetic transcription; genetically modified animals; host organism interaction; immunogenetics; interferon inducers; interferons; laboratory mouse; microarray technology; microorganism immunology; parainfluenza virus type 1; phenotype; toll like receptor; virus diseases; yeast two hybrid system

We do not yet fully understand how host cells recognize and respond to viral infections, and our broad goal is to1 clarify mechanisms of anti-viral innate immunity in mammals. We have previously studied microbial components recognized by Toll-like receptors (TLRs) and established mouse models lacking each TLR. These studies revealed that viruses are recognized by TLR-dependent and -independent mechanisms and that recognition leads to induction of type I interferons (IFNs), cytokines critical for antiviral host defense in mammals. Although TLR signaling pathways leading to IFN production have been extensively studied, the molecular mechanisms of TLR-independent viral detection have remained poorly understood. Recently, we have focused on a family of potential cytoplasmic viral detectors and their signaling intermediates. We also have reason to believe that IFN-independent anti-viral host defense mechanisms operate in mammals, although we do not know precisely what these mechanisms might entail. In the present proposal, we plan to utilize mouse reverse genetics (gene targeting) to generate and analyzing mutant mice lacking molecules that have a strong likelihood of participating in antiviral responses. Combining targeted mutations with one another and using classical methods of pathway analysis, we hope to understand the how the presence of viruses is sensed, how signals are initiated, and how they are transduced to yield a defensive response. First, we will continue our studies of potential intracellular viral detectors and their signaling molecules leading to the production of type I IFNs and proinflammatory cytokines. Second, we will search for new candidate molecules potentially involved in signaling pathways that lead from from cytoplasmic viral detectors to permit type I IFN production. Toward this end, we will use expression cloning and yeast two-hybrid screening. Third, we will try to understand and characterize novel anti-viral host defense mechanisms. To do so we will rely upon data from the forward genetic initiatives pursued in Project 1 and Project 2. We will generate mice with mutations in homologues of candidate molecules identified by Drosophila forward genetics conducted by the Strasbourg group. We will also target paralogues of molecules identified by mouse forward genetics conducted by the La Jolla group. By comparing the phenotypes of these mice with those bearing mutations affecting pathways serving the TLRs or cytoplasmic viral detectors, we will develop a comprehensive understanding of the host response. The successful attainment of such an understanding is enormously important, since viral infections are one of the most important causes of death worldwide. Potentially, the new concepts that we develop may be applied to the improvement of anti-viral vaccines and to the development of small molecules that enforce anti-viral responses through interaction with newly identified molecules.

我们尚未完全了解宿主细胞如何识别和应对病毒感染，我们的广泛目标是 TO1澄清哺乳动物抗病毒先天免疫的机制。我们以前已经研究了微生物 由Toll样受体（TLR）识别的组件和缺乏每个TLR的老鼠模型。 这些研究表明，病毒是通过TLR依赖性和非依赖性机制识别的，并且 这种识别导致诱导I型干扰素（IFNS），细胞因子对抗病毒宿主防御至关重要 哺乳动物。尽管已广泛研究了导致IFN产生的TLR信号通路，但 TLR非依赖性病毒检测的分子机制尚不清楚。最近，我们 专注于潜在的细胞质病毒探测器及其信号中间体的家族。我们也是 有理由相信IFN独立的抗病毒宿主防御机制在哺乳动物中起作用， 尽管我们不确定这些机制可能需要什么。在本提案中，我们计划 利用小鼠反向遗传学（基因靶向）来产生和分析缺乏分子的突变小鼠 参与抗病毒反应的可能性很大。将目标突变相结合 并使用经典的途径分析方法，我们希望了解病毒的存在如何 感觉到信号的启动方式以及如何转导以产生防御响应。首先，我们会的 继续我们对潜在的细胞内病毒探测器及其信号分子的研究 I型IFN和促炎细胞因子的产生。其次，我们将搜索新的候选分子 可能参与从细胞质病毒探测器导致的信号通路，以允许I型IFN 生产。为此，我们将使用表达克隆和酵母双杂交筛选。第三，我们会尝试 了解和表征新型的抗病毒宿主防御机制。为此，我们将依靠来自 项目1和项目2中采用的远期遗传倡议。我们将产生具有突变的小鼠 由果蝇前遗传学鉴定的候选分子的同源物由Strasbourg进行 团体。我们还将靶向由LA进行的小鼠正向遗传学鉴定的分子的旁系同源物 Jolla集团。通过将这些小鼠的表型与影响途径的轴承突变进行比较 为TLR或细胞质病毒探测器服务，我们将对宿主产生全面的理解 回复。由于病毒感染，成功获得这种理解非常重要 是全球死亡最重要的原因之一。可能，我们开发的新概念可能 应用于改善抗病毒疫苗和强制执行的小分子的发展 抗病毒反应通过与新鉴定的分子相互作用而产生。